Total joint replacements are commonly performed surgeries that reduce pain and improve quality of life. Indeed, every year nearly 800,000 patients will undergo hip or knee replacement surgeries in the United States (NIAMS website. 2012). The failure rate of hip and knee prostheses is approximately 1%/year although younger age is an important risk factor for prosthesis failure requiring revision arthroplasty (Ong, et al., Clin Orthop Relat Res, 2010, 468(11): 3070-6). The most common cause for prosthesis failure requiring revision is peri-implant osteolysis leading to loosening of the prosthesis. As joints are replaced in younger individuals and the elderly live longer and lead more active lives it is imperative that new approaches to bone preservation be developed to prevent a growing wave of revision surgeries with their associated morbidities, mortality and cost.
Osteolysis results from the inflammatory response to wear particles (ultra high molecular weight polyethylene particles, UHMWPE) or metal fragments leading to stimulation of osteoclast differentiation and bone resorption. In recent studies, using a murine model of wear particle-induced osteolysis, stimulation of adenosine A2A receptors (A2A receptor) dramatically reduced wear particle-induced bone resorption by diminishing M-CSF, RANKL, IL-1 and TNFα and increasing IL-10 levels in the exudate overlying the implanted wear particles and by diminishing the number of cells expressing TNFα, RANK, CD68, αSMA, RANKL and osteopontin while increasing the number of osteoprotegerin-expressing cells in the calvarial bone (Mediero, et al., Sci Transl Med, 2012; 4(135): 135-65). As had previously been reported, not only does the inflammatory response to UHMWPE promote a marked increase in osteoclasts but a reduction in osteoblasts as well leading to uncoupling of bone resorption from bone formation that characterizes bone homeostasis.
Although cytokine-stimulated osteoclast activation has been well described as the principal pathophysiologic mechanism mediating inflammatory bone loss there is diminished bone formation and the mechanism for this has been less well studied. Secretion of inhibitors of the Wnt-frizzled pathway, such as DKK-1, at inflamed sites is thought to inhibit osteoblast differentiation and function (Reviewed in Walsh, et al., Immunol Rev, 2010; 233(1):301-12). More recently axonal guidance proteins, such as semaphorin 4D (sema4D), produced by osteoclasts and activated macrophages have been reported to diminish osteoblast differentiation and function (Negishi-Koga, et al., Nature Medicine, 2011; 17(11): 1473-80). The role of other axonal guidance proteins in coupling or uncoupling of bone resorption and synthesis have also become a subject of interest (Hughes, A., et al., Calcified Tissue International, 2012; 90(2): 151-62; Hayashi, et al., Nature, 2012; 485(7396): 69-74; Sutton, et al., Molecular Endocrinology, 2008; 22(6): 1370-81; Tamagnone, et al., Nat Cell Biol, 2006, 8(6): 545-7; Koh, et al., J Hum Genet, 2006; 51(2): 112-7 and Togari, et al., Brain Research, 2000; 878(1-2): 204-9). In the murine calvaria model of wear particle-induced bone resorption, A2A receptor agonists were shown to inhibit the accumulation of sema4D-expressing cells, most of which are CD68+ macrophages, at osteolytic sites. Although the expression of sema4D on T cells and other cells has been well documented there is little evidence that macrophages express this molecule (Giordano, et al., Nat Cell Biol, 2002; 4(9): 720-4 and Sierra, et al., J Exp Med, 2008; 205(7): 1673-85).
Axon guidance (also called axon path finding) is a subfield of neural development concerning the process by which neurons send out axons to reach the correct targets. Axons often follow very precise paths in the nervous system, and how they manage to find their way so accurately is being researched. Growing axons have a highly motile structure at the growing tip called the growth cone, which “sniffs out” the extracellular environment for signals that instruct the axon which direction to grow. These signals, called guidance cues, can be fixed in place or diffusible; they can attract or repel axons. Growth cones contain receptors that recognize these guidance cues and interpret the signal into a chemotropic response. The general theoretical framework is that when a growth cone “senses” a guidance cue, the receptors activate various signaling molecules in the growth cone that eventually affect the cytoskeleton. If the growth cone senses a gradient of guidance cue, the intracellular signaling in the growth cone happens asymmetrically, so that cytoskeletal changes happen asymmetrically and the growth cone turns toward or away from the guidance cue.
A combination of genetic and biochemical methods (see below) has led to the discovery of several important classes of axon guidance molecules and their receptors. Netrins are secreted molecules that can act to attract or repel axons by binding to their receptors, DCC and UNC5. Slits also known as Sli are secreted proteins that normally repel growth cones by engaging Robo (Roundabout) class receptors. Ephrins are cell surface molecules that activate Eph receptors on the surface of other cells. This interaction can be attractive or repulsive. In some cases, Ephrins can also act as receptors by transducing a signal into the expressing cell, while Ephs act as the ligands. Signaling into both the Ephrin- and Eph-bearing cells is called “bi-directional signaling.” Semaphorins occur as many types and are primarily axonal repellents, and activate complexes of cell-surface receptors called Plexins and Neuropilins. Cell adhesion molecules (CAMs) are integral membrane proteins mediating adhesion between growing axons and eliciting intracellular signalling within the growth cone. CAMs are the major class of proteins mediating correct axonal navigation of axons growing on axons (fasciculation). There are two CAM subgroups: IgSF-CAMs (belonging to the immunoglobulin superfamily) and Cadherins (Ca-dependent CAMs).
In addition, many other classes of extracellular molecules are used by growth cones to navigate properly including developmental morphogens, such as BMPs, Wnts, Hedgehog, and FGFs, extracellular matrix and adhesion molecules such as laminin, tenascins, proteoglycans, N-CAM, and L1, growth factors like NGF, and neurotransmitters and modulators like GABA.
Growing axons rely on variety of guidance cues in deciding upon a growth pathway. The growth cones of extending axons process these cues in an intricate system of signal interpretation and integration, in order to insure appropriate guidance. Adhesive cues provide physical interaction with the substrate necessary for axon protrusion. These cues can be expressed on glial and neuronal cells the growing axon contacts or be part of the extracellular matrix. Examples are laminin or fibronectin, in the extracellular matrix, and cadherins or Ig-family cell-adhesion molecules, found on cell surfaces. Tropic cues act as attractants or repellents and cause changes in growth cone motility by acting on the cytoskeleton through intracellular signaling. For example, Netrin plays a role in guiding axons through the midline, acting as both an attractant and a repellent. While Semaphorin3A, helps axons grow from the olfactory epithelium to map different locations in the olfactory bulb. Modulatory cues influence the sensitivity of growth cones to certain guidance cues. For instance, neurotrophins can make axons less sensitive to the repellent action of Semaphorin3A.
Given the abundance of these different guidance cues it was previously believed that growth cones integrate various information by simply summing the gradient of cues, in different valences, at a given point in time, to making a decision on the direction of growth. However, studies in vertebrate nervous systems of ventral midline crossing axons, has shown that modulatory cues play a crucial part in tuning axon responses to other cues, suggesting that the process of axon guidance is nonlinear. For examples, commisurial axons are attracted by netrin and repelled by slit. However, as axons approach the midline, the repellent action of Slit is suppressed by Robo-3/Rig-1 receptor. Once the axons cross the midline, activation of Robo by Slit silences Netrin-mediated attraction, and the axons are repelled by Slit.
The netrin family is composed mostly of secreted proteins which serve as bifunctional signals: attracting some neurons while repelling others during the development of brain. Expressed in the midline of all animals possessing bilateral symmetry, they can act as long or short range signals during neurogenesis. In order to carry out their functions, netrins interact with specific receptors: DCC or UNC-5 depending on whether they are trying to attract or repel neurons respectively. There is a high degree of conservation in the secondary structure of netrins, which has several domains which are homologous with laminin at the amino terminal end. The C-terminal domain is where most of the variation is found between species and contains different amino acids which allow interaction with specific proteins in extracellular matrix or on cell surface. The differences in terms of structure and function have led to the identifications of several different types of netrins including netrin-1, netrin-3, and netrins-G.
Netrin-1 is found in the floor plate and neuroepithelial cells of the ventral region of the spinal cord, as well as other locations in the nervous system including the somatic mesoderm, pancreas and cardiac muscle. Its main role is in axonal guidance, neuronal migration and morphogenesis of different branching structures. Mice with mutations in the netrin-1 gene were observed to be lacking in forebrain and spinal cord commissural axons. Netrin-3 is different from other netrins. While expressed during development of the peripheral nervous system in the motor, sensory and sympathetic neurons, it is very limited in the central nervous system. Studies with netrin-3 have noticed a reduced ability to bind with DCC receptors when compared with netrin-1. This suggests that it mainly operates through other receptors. Netrins-G are secreted but remain bound to the extracellular surface of the cell membrane through Glycophosphatidylinositol (GPI). They are expressed predominantly in the central nervous system in places such as the thalamus and mitral cells of the olfactory bulb.[7] They do not bind to DCC or UNC-5 and instead bind to ligand NGL-1, which results in an intracellular transduction cascade. The two versions, netrins-G1 and netrins-G2, are found only in vertebrates. It is believed that they evolved independently of other netrins in order to facilitate the construction of the brain.
DCC and UNC-5 proteins carefully mediate netrin-1 responses. The UNC-5 protein is mainly involved in signaling repulsion. DCC, which is implicated in attraction, can also serve as a co-factor in repulsion signaling when far away from the source of netrin-1. DCC is highly expressed in the central nervous system and associated with the basal lamina of epithelia cells. In the absence of netrin-1, these receptors are known to induce apoptosis.